In processing cocoa beans the generation of the typical cocoa flavor requires two steps, the fermentation step and the roasting step. During fermentation the pulp surrounding the beans is degraded by micro-organisms with the sugars contained in the pulp being essentially transformed to acids. Fermentation also results in a release of peptides exhibiting differing sizes and a generation of a high level of free hydrophobic amino acids. This latter finding led to the hypothesis that proteolysis occurring during fermentation is not due to a random protein hydrolysis but seems to be rather based on the activity of specific endoproteinases.
This specific mixture of peptides and hydrophobic amino acids is deemed to represent cocoa-specific flavor precursors. During the second step of cocoa flavor production, the roasting step, the oligopeptides and amino acids generated at the stage of fermentation obviously undergo a Maillard reaction with reducing sugars present eventually yielding the substances responsible for the cocoa flavor as such.
So far, research has tried to uncover the molecular pathway of producing cocoa flavor precursors in characterizing enzymes involved in said process and the relevant polypeptide (s), from which the peptides and/or free amino acids are produced.
As for the enzymes many different endo-and exoproteinase activities have been found to participate in the production of cocoa flavor precursors, such as an aspartic endoproteinase activity (Voigt et al., J. Plant Physiol. 145 (1995), 299-307), which accumulates with the vicilin-class (7S) globulin during bean ripening or a cysteine endoproteinase activity, which increases during the germination process when degradation of globular storage protein increases during the germination process when degradation of globular storage protein occurs (Biehl et al., Cocoa Research Conference, Salvador, Bahia, Brasil, Nov. 17-23, 1996).
Moreover, a carboxypeptidase activity has been identified which preferentially splits hydrophobic amino acids from the carboxy-terminus of peptides.
Apart from the enzymes also the protein source of the peptides/amino acids seems to be of importance for the generation of cocoa flavor precursors.
During cocoa bean fermentation the percentage reduction of protein concentration observed for vicilin and albumin was 88.8% and 47.4%, respectively (Amin et al. J. Sci. Food Agric. 76 (1998), 123-128). When peptides obtained by proteolysis of the globulin (vicilin) fraction were post-treated with carboxypeptidase, preferentially hydrophobic amino acids were released and a typical cocoa aroma was detected after roasting in the presence of reducing sugars (Voigt et al., Food Chem. 50 (1994), 177-184). Contrary to that, the predominant amino acids released from the albumin-derived peptides were aspartic acid, glutamic acid and asparagine and no cocoa aroma could be detected. It was therefore concluded that cocoa-specific aroma precursors are mainly derived from the vicilin-like globulins of cocoa, which constitute more than 30% of the total protein contents in the mature cocoa seed. Consequently, the mixture of hydrophobic free amino acids and remaining oligopeptides required for the generation of the typical cocoa flavor components seem to be determined by the particular structure of the cocoa vicilin-class globulins.
Although it is known that hydrophobic amino acids are important cocoa flavor precursors, the specific peptides responsible for generating cocoa flavor during roasting remain by and large un-characterized. Consequently, there is a need in the art to provide further structural data of such peptides and of the way they are produced from their original proteinaceous source in order to be capable to use those peptides for the production of a well-balanced cocoa and/or chocolate flavor.
In WO 91/19801 two major cocoa seed storage proteins and the DNA sequences encoding said polypeptides are disclosed. These two proteins exhibit a molecular weight of 47 kDa and 31 kDa, respectively, and seem to be the vicilin polypeptides, which are deemed to be the source of the flavoring peptides creating the characteristic cocoa flavor. Though the polypeptides have recombinantly been provided as such no specific data as to the synthesis of the flavoring peptides have been provided.
Consequently, there is a need for further elucidating the generation of cocoa flavor from the relevant protein material contained in cocoa.
In order to solve the above problem research has naturally focused on the vicilin polypeptides in cocoa beans, since other protein material contained therein was not considered to contribute to the generation of cocoa flavor as such. In contrast to this general belief the present inventors have now surprisingly found that a polypeptide, being a member of the albumin family, also contributes to the characteristic cocoa flavor during fermentation and roasting.